370 research outputs found
BRCA to the future: towards best testing practice in the era of personalised healthcare
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Detection of BRCA1/2 large genomic rearrangements in breast and ovarian cancer patients: an overview of the current methods
No full textIntroduction: Currently, genetic testing of BRCA1/2 genes includes screening for single-nucleotide variants, small insertions/deletions, and copy number variations (CNVs). In fact, many studies document the involvement of BRCA1/2 gene rearrangements in genetic predisposition to breast and ovarian cancer. Large genomic rearrangements (LGRs) of BRCA1 may account for up to one-third of all disease-causing alterations in various populations, while LGRs in BRCA2 are less frequently observed. Areas covered: We aimed to present an overview of current technologies employed in molecular diagnosis of BRCA1/2 LGRs. The most relevant literature papers, showing the application of new strategies, were considered. Expert opinion: Currently, the progress of next-generation sequencing (NGS) technologies allows for the validation of new pipelines able to provide rapid and effective results, ensuring the sensitivity and specificity requested for the detection of BRCA1/2 LGRs. Multiplex ligation-dependent probe amplification remains the gold standard to confirm NGS CNVs results and to perform fast screening in families where a pathogenic rearrangement has been detected in a proband
Circulating fetal cell-free DNA and prenatal molecular diagnostics: are we ready for consensus?
No full textAbstract Circulating fetal cell-free DNA (cffDNA) is emerging as the most reliable known target for prenatal molecular diagnostics. Different points of view are expressed in literature regarding the safe use of cffDNA for all types of molecular tests, above all those used to detect maternal DNA rather than fetal mutation. The aim of the present study was therefore to achieve consensus on guidelines conducive to standardizing current procedures, which differ between laboratories, and to design stringent technical protocols for the analysis of cffDNA
Is quantitative real time polymerase chain reaction MCAM transcript assay really suitable for prognostic and predictive management of melanoma patients?
No full textBackground: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients.
Methods: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results.
Results: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894in5TTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified.
Conclusion: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing. (C) 2014 Elsevier B.V. All rights reserved
Contribution of the TA repeats on melting temperature (T(m)) in a double strand DNA: Comparison of two methods and implications in molecular diagnostics
No full textThe present paper is aimed to discuss and revise a not recent molecular laboratory protocol describing the behavior of UGT1A1 gene melting curves. Since some problems in the standardization of this already published molecular protocol were found, we tried to improve this method in order to correctly set up the amplification and melting profile steps. Under our standardized conditions, we were able to perfectly distinguish the three different UGT1A1 genotypes since each one showed a peculiar melting behavior, as compared with those previously published by another group. Finally, some suggestions and indications are provided for laboratory specialists interested in Gilbert syndrome diagnostics. Based on these results, we underline the need of more standardized protocols above all when they are used for clinical diagnostics. © 2011
Il deficit di G6PD in Medicina di Laboratorio = G6PD deficiency in Laboratory Medicine
No full textGlucose 6-phosphate dehydrogenase has a key role in the production of the reducing power necessary to face oxidative stress and for providing ribose, which is a basic constituent of nucleic acids. It is therefore not surprising that the gene is present in all organisms, and that is also highly conserved during the evolution. The occurrence of G6PD mutants may have important consequences in carriers, depending on the class of the mutants, triggering events and various comorbidities. Acute and chronic hemolytic anemias are the most typical clinical hallmarks. The determination and characterization of G6PD can be achieved by qualitative tests, various quantitative catalytic activity determinations, and by molecular biology techniques. Sample collection and stability is not a critical problem, but some pre-analytical conditions in vivo(previous transfusions, recent blood losses) should be known by the laboratory before measuring G6PD. A number of quantitative methods is available and there is no consensus on the measuring temperature (30 or 37°C) and on the reference ranges. The consequence is that the state-of-the-art of the various methods is variable, as clearly proven from recent EQAS analyses. Moreover, analytical goals have not been defined yet. In conclusion, we believe that this test needs better attention from laboratory professionals in order to offer an improved service to patients with G6PD deficiency anemias and related complications
Could G6PD-Buenos-Aires confirm the existence of the "structural NADP+ binding site" and its strategic role for the stability and/or activity enzyme?
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The friendly use of chloroquine in the COVID-19 disease: A warning for the G6PD-deficient males and for the unaware carriers of pathogenic alterations of the G6PD gene
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